Three NO synthases (iNOS, nNOS, and eNOS) function broadly in human health and disease. Our goal is to define the mechanism of NO synthesis and the structure-function aspects of NOS catalysis, which should help to develop clinical strategies to control NO availability. All NOS contain heme and flavins and catalyze a two-step oxidation of L-arginine to make NO. NOSs are unique among flavoheme enzymes because they contain 6R-tetrahydrobiopterin (HUB) as a bound cofactor. Moreover, their H4B cofactor undergoes novel one- electron transitions during catalysis. Our previous studies showed how H4B radical formation relates to steps in oxygen activation and catalysis in the three NOS, and probed some of the structure-function aspects of H4B redox function. Our current Aims describe biochemical, kinetic, molecular biological, and biophysical studies that will advance our understanding of NOS catalysis by further defining the mechanisms, regulation, and impact of H4B functions. Aim 1. Investigate the structural, thermodynamic, and NOS isozyme-specific basis for regulating H4B radical formation. H4B transfers an electron to a heme-dioxy intermediate in both reactions of NO synthesis (Arg hydroxylation and N-hydroxyArg oxidation). We hypothesize that the kinetics and extent of H4B radical formation are tuned by surrounding protein residues and by the pterin ring structure itself. We will test the function of specific NOS residues and pterin structural analogs, and investigate thermodynamic control of H4B radical formation in NOS. Aim 2. Investigate redox-independent effects of pterins on the reduction, stability, and subsequent reactivity of the FeII02 intermediate in NOS. We will utilize redox-inactive pterins to investigate how bound pterins impact NOS flavoprotein electron transfer to the heme, the stability of the FeIIO2 intermediate, and the further reactivity of NOS heme-oxy species to generate products from Arg and NONA. This work will provide a comprehensive view of how pterin influences NOS catalysis. Aim 3. Investigate the reductive transitions of the H4B radical and how they are regulated. The H4B radical formed in both reactions of NO synthesis must be reduced back to H4B before the enzyme can continue catalysis. How this occurs is unclear. We hypothesize that the H4B radical is reduced by distinct mechanisms in each reaction of NO synthesis. We have developed methods to monitor H4B radical reduction in single catalytic turnover reactions and will examine the mechanistic and regulatory aspects. Aim 4. Initiate 13C, 15N, and HSQC NMR studies of bound H4B in NOS. We hypothesize that the NOS protein creates an electronic environment for H4B that favors its one-electron redox transitions. We will perform NMR studies on 13C- and 15N-enriched H4B bound in NOS or its mutants to test specific hypotheses derived from our protein crystal structures. Relevance: By clarifying how nitric oxide production occurs and is regulated, our work may help to develop treatments for human diseases that involve making too much or too little nitric oxide.